The deubiquitinases USP33 and USP20 coordinate β2 adrenergic receptor recycling and resensitization

Agonist‐induced ubiquitination of the β₂ adrenergic receptor (β₂AR) functions as an important post‐translational modification to sort internalized receptors to the lysosomes for degradation. We now show that this ubiquitination is reversed by two deubiquitinating enzymes, ubiquitin‐specific proteases (USPs) 20 and 33, thus, inhibiting lysosomal trafficking when concomitantly promoting receptor recycling from the late‐endosomal compartments as well as resensitization of recycled receptors at the cell surface. Dissociation of constitutively bound endogenously expressed USPs 20 and 33 from the β₂AR immediately after agonist stimulation and reassociation on prolonged agonist treatment allows receptors to first become ubiquitinated and then deubiquitinated, thus, providing a ‘trip switch’ between degradative and recycling pathways at the late‐endosomal compartments. Thus, USPs 20 and 33 serve as novel regulators that dictate both post‐endocytic sorting as well as the intensity and extent of β₂AR signalling from the cell surface.     

Chronic increases in sphingosine kinase-1 activity induce a pro-inflammatory, pro-angiogenic phenotype in endothelial cells

Sphingosine kinase-1 (SK1) promotes the formation of sphingosine-1-phosphate (S1P), which has potent pro-inflammatory and pro-angiogenic effects. We investigated the effects of raised SK1 levels on endothelial cell function and the possibility that this signaling pathway is activated in rheumatoid arthritis. Human umbilical vein endothelial cells with 3- to 5-fold SK1 (EC^SK) overexpression were generated by adenoviral and retroviralmediated gene delivery. The activation state of these cells and their ability to undergo angiogenesis was determined. S1P was measured in synovial fluid from patients with RA and OA. EC^SK showed an enhanced migratory capacity and a stimulated rate of capillary tube formation. The cells showed constitutive activation as evidenced by the induction of basal VCAM-1 expression, and further showed a more augmented VCAM-1 and E selectin response to TNF compared with empty vector control cells (EC^EV). These changes had functional consequences in terms of enhanced neutrophil binding in the basal and TNFstimulated states in EC^SK. By contrast, over-expression of a dominant-negative SK inhibited the TNF-induced VCAM-1 and E selectin and inhibited PMN adhesion, confirming that the observed effects were specifically mediated by SK. The synovial fluid levels of S1P were significantly higher in patients with RA than in those with OA. Small chronic increases in SK1 activity in the endothelial cells enhance the ability of the cells to support inflammation and undergo angiogenesis, and sensitize the cells to inflammatory cytokines. The SK1 signaling pathway is activated in RA, suggesting that manipulation of SK1 activity in diseases of aberrant inflammation and angiogenesis may be beneficial.     

CGI-58/ABHD5 is a coenzyme A-dependent lysophosphatidic acid acyltransferase

Mutations in human CGI-58/ABHD5 cause Chanarin-Dorfman syndrome (CDS), characterized by excessive storage of triacylglycerol in tissues. CGI-58 is an α/β-hydrolase fold enzyme expressed in all vertebrates. The carboxyl terminus includes a highly conserved consensus sequence (HXXXXD) for acyltransferase activity. Mouse CGI-58 was expressed in Escherichia coli as a fusion protein with two amino terminal 6-histidine tags. Recombinant CGI-58 displayed acyl-CoA-dependent acyltransferase activity to lysophosphatidic acid, but not to other lysophospholipid or neutral glycerolipid acceptors. Production of phosphatidic acid increased with time and increasing concentrations of recombinant CGI-58 and was optimal between pH 7.0 and 8.5. The enzyme showed saturation kinetics with respect to 1-oleoyl-lysophosphatidic acid and oleoyl-CoA and preference for arachidonoyl-CoA and oleoyl-CoA. The enzyme showed slight preference for 1-oleoyl lysophosphatidic acid over 1-palmitoyl, 1-stearoyl, or 1-arachidonoyl lysophosphatidic acid. Recombinant CGI-58 showed intrinsic fluorescence for tryptophan that was quenched by the addition of 1-oleoyl-lysophosphatidic acid, oleoyl-CoA, arachidonoyl-CoA, and palmitoyl-CoA, but not by lysophosphatidyl choline. Expression of CGI-58 in fibroblasts from humans with CDS increased the incorporation of radiolabeled fatty acids released from the lipolysis of stored triacylglycerols into phospholipids. CGI-58 is a CoA-dependent lysophosphatidic acid acyltransferase that channels fatty acids released from the hydrolysis of stored triacylglycerols into phospholipids.     

Immobilization of acidic lipase derived from Pseudomonas gessardii onto mesoporous activated carbon for the hydrolysis of olive oil

Mesoporous activated carbon (MAC) derived from rice husk is used for the immobilization of acidic lipase (ALIP) produced from Pseudomonas gessardii. The purified acidic lipase had the specific activity and molecular weight of 1473 U/mg and 94 kDa respectively. To determine the optimum conditions for the immobilization of lipase onto MAC, the experiments were carried out by varying the time (10–180 min), pH (2–8), temperature (10–50 °C) and the initial lipase activity (49 × 10³, 98 × 10³, 147 × 10³ and 196 × 10³ U/l in acetate buffer). The optimum conditions for immobilization of acidic lipase were found to be: time—120 min; pH 3.5; temperature—30 °C, which resulted in achieving a maximum immobilization of 1834 U/g. The thermal stability of the immobilized lipase was comparatively higher than that in its free form. The free and immobilized enzyme kinetic parameters (K_m and V_max) were found using Michaelis–Menten enzyme kinetics. The K_m values for free enzyme and immobilized one were 0.655 and 0.243 mM respectively. The immobilization of acidic lipase onto MAC was confirmed using Fourier Transform-Infrared Spectroscopy, X-ray diffraction analysis and scanning electron microscopy.     

The SbcCD complex of Deinococcus radiodurans contributes to radioresistance and DNA strand break repair in vivo and exhibits Mre11–Rad50 type activity in vitro

Deinococcus radiodurans lacks a homologue of the recB and recC genes, and the sbcA/B genes, of Escherichia coli. Thus, DNA strand break repair in Deinococcus proceeds by pathways that do not utilize these proteins. Unlike E. coli, the absence of recBC and sbcA/sbcB, and presence of only sbcC and sbcD in Deinococcus, indicates an enigmatic role of SbcCD in this bacterium. Studies on sbcCD mutation in Deinococcus showed nearly a 100-fold increase in gamma radiation sensitivity as compared to wild type. The mutant showed a higher rate of in vivo DNA degradation during the post-irradiation recovery period that corresponds to the RecA-dependent DSB repair phase. These cells showed a typical NotI pattern of DNA reassembly during the early phase of DSB repair, but were defective for the subsequent RecA-dependent phase II of DSB repair. Hydrogen peroxide had no effect on cell survival of the mutant. While its tolerance to higher doses of UVC and mitomycin C was significantly decreased as compared to wild type. Purified recombinant SbcCD proteins showed single-stranded endonuclease and 3′ → 5′ double-stranded DNA exonuclease activities similar to that of the Mre11–Rad50 complex, which is required for DNA strand break repair in higher organisms. These results suggested that the Mre11–Rad50 type nuclease activity of SbcCD proteins contributes to the radiation resistance of D. radiodurans perhaps by promoting the RecA-dependent DSB repair required for polyploid genome maturation.     

A Safe Vaccine (DV-STM-07) against Salmonella Infection Prevents Abortion and Confers Protective Immunity to the Pregnant and New Born Mice

Pregnancy is a transient immuno-compromised condition which has evolved to avoid the immune rejection of the fetus by the maternal immune system. The altered immune response of the pregnant female leads to increased susceptibility to invading pathogens, resulting in abortion and congenital defects of the fetus and a subnormal response to vaccination. Active vaccination during pregnancy may lead to abortion induced by heightened cell mediated immune response. In this study, we have administered the highly attenuated vaccine strain ΔpmrG-HM-D (DV-STM-07) in female mice before the onset of pregnancy and followed the immune reaction against challenge with virulent S. Typhimurium in pregnant mice. Here we demonstrate that DV-STM-07 vaccine gives protection against Salmonella in pregnant mice and also prevents Salmonella induced abortion. This protection is conferred by directing the immune response towards Th2 activation and Th1 suppression. The low Th1 response prevents abortion. The use of live attenuated vaccine just before pregnancy carries the risk of transmission to the fetus. We have shown that this vaccine is safe as the vaccine strain is quickly eliminated from the mother and is not transmitted to the fetus. This vaccine also confers immunity to the new born mice of vaccinated mothers. Since there is no evidence of the vaccine candidate reaching the new born mice, we hypothesize that it may be due to trans-colostral transfer of protective anti-Salmonella antibodies. These results suggest that our vaccine DV-STM-07 can be very useful in preventing abortion in the pregnant individuals and confer immunity to the new born. Since there are no such vaccine candidates which can be given to the new born and to the pregnant women, this vaccine holds a very bright future to combat Salmonella induced pregnancy loss.     

Axonal transport of microtubules: the long and short of it

Recent studies on cultured neurons have demonstrated that microtubules are transported down the axon in the form of short polymers. The transport of these microtubules is bidirectional, intermittent, asynchronous, and occurs at the fast rate of known motors. The majority of the microtubule mass in the axon exists in the form of longer immobile microtubules. We have proposed a model called 'cut and run', in which the longer microtubules are mobilized by enzymes that sever them into shorter mobile polymers. In this view, the molecular motors that transport microtubules are not selective for short microtubules but rather impinge upon microtubules irrespective of their length. In the case of the longer microtubules, these motor-driven forces do not transport the microtubules in a rapid and concerted fashion but presumably affect them nonetheless. Here, we discuss the mechanisms by which the short microtubules are transported and suggest possibilities for how analogous mechanisms may align and organize the longer microtubules and functionally integrate them with each other and with the actin cytoskeleton.     

Antagonistic forces generated by cytoplasmic dynein and myosin-II during growth cone turning and axonal retraction

Cytoplasmic dynein transports short microtubules down the axon in part by pushing against the actin cytoskeleton. Recent studies have suggested that comparable dynein-driven forces may impinge upon the longer microtubules within the axon. Here, we examined a potential role for these forces on axonal retraction and growth cone turning in neurons partially depleted of dynein heavy chain (DHC) by small interfering RNA. While DHC-depleted axons grew at normal rates, they retracted far more robustly in response to donors of nitric oxide than control axons, and their growth cones failed to efficiently turn in response to substrate borders. Live cell imaging of dynamic microtubule tips showed that microtubules in DHC-depleted growth cones were largely confined to the central zone, with very few extending into filopodia. Even under conditions of suppressed microtubule dynamics, DHC depletion impaired the capacity of microtubules to advance into the peripheral zone of the growth cone, indicating a direct role for dynein-driven forces on the distribution of the microtubules. These effects were all reversed by inhibition of myosin-II forces, which are known to underlie the retrograde flow of actin in the growth cone and the contractility of the cortical actin during axonal retraction. Our results are consistent with a model whereby dynein-driven forces enable microtubules to overcome myosin-II-driven forces, both in the axonal shaft and within the growth cone. These dynein-driven forces oppose the tendency of the axon to retract and permit microtubules to advance into the peripheral zone of the growth cone so that they can invade filopodia.     

Perilipin A mediates the reversible binding of CGI-58 to lipid droplets in 3T3-L1 adipocytes

Perilipins, the major structural proteins coating the surfaces of mature lipid droplets of adipocytes, play an important role in the regulation of triacylglycerol storage and hydrolysis. We have used proteomic analysis to identify CGI-58, a member of the alpha/beta-hydrolase fold family of enzymes, as a component of lipid droplets of 3T3-L1 adipocytes. CGI-58 mRNA is highly expressed in adipose tissue and testes, tissues that also express perilipins, and at lower levels in liver, skin, kidney, and heart. Both endogenous CGI-58 and an ectopic CGI-58-GFP chimera show diffuse cytoplasmic localization in 3T3-L1 preadipocytes, but localize almost exclusively to the surfaces of lipid droplets in differentiated 3T3-L1 adipocytes. The localization of endogenous CGI-58 was investigated in 3T3-L1 cells stably expressing mutated forms of perilipin using microscopy. CGI-58 binds to lipid droplets coated with perilipin A or mutated forms of perilipin with an intact C-terminal sequence from amino acid 382 to 429, but not to lipid droplets coated with perilipin B or mutated perilipin A lacking this sequence. Immunoprecipitation studies confirmed these findings, but also showed co-precipitation of perilipin B and CGI-58. Remarkably, activation of cAMP-dependent protein kinase by the incubation of 3T3-L1 adipocytes with isoproterenol and isobutylmethylxanthine disperses CGI-58 from the surfaces of lipid droplets to a cytoplasmic distribution. This shift in subcellular localization can be reversed by the addition of propanolol to the culture medium. Thus, CGI-58 binds to perilipin A-coated lipid droplets in a manner that is dependent upon the metabolic status of the adipocyte and the activity of cAMP-dependent protein kinase.